Method for activating peroxisome proliferator activated receptor-gamma

ABSTRACT

The invention relates to a method of identifying nuclear receptor controlled genes in specific tissues. In particular, the method also provides a method of activating PPARγ nuclear receptor controlled target genes in vivo in a tissue-specific manner.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of co-pending application Ser.No. 09/778,032, filed Feb. 7, 2001, which is a continuation-in-part ofco-pending prior application Ser. No. 09/596,083, filed Jun. 16, 2000,which claims priority from provisional application Ser. No. 60/139,718,filed Jun. 18, 1999.

BACKGROUND OF THE INVENTION

[0002] 1. Technical Field

[0003] This application relates to a method of activating PPARγ nuclearreceptor controlled target genes in vivo. Activation of PPARγ in certaintissues is sufficient to prevent the development of diabetes.

[0004] 2. Background of the Invention

[0005] The thiazolidinedione class of antidiabetic drugs represent oneof the few treatments of diabetes that alleviate insulin resistance,hyperglycemia and hyperlipidemia in patients with NIDDM.Thiazolidinediones are ligands for peroxisome proliferator activatedreceptor-γ (PPARγ), a member of the nuclear receptor superfamily. Thismolecular linkage implies that thiazolidinediones achieve their insulinresistance effects by regulating PPARγ target genes. However, theprecise pathway connecting PPARγ activation to insulin sensitizationremains a mystery. In particular, the target tissue for PPARγ action isunknown. Therefore, it is unclear which PPARγ target genes contribute tothe normalization of insulin response.

[0006] Molecular cloning studies have demonstrated that nuclearreceptors for steroid, retinoid, vitamin D and thyroid hormones comprisea superfamily of regulatory proteins that are structurally andfunctionally related. Nuclear receptors contain a central DNA bindingdomain that binds to cis-acting elements (response elements) in thepromoters of their target genes. Once bound to a response element,nuclear receptors activate transcription of specific genes through theirconserved C-terminal ligand binding domains which bind hormones withhigh affinity and specificity. The ligand binding domain is a complexentity containing several embedded subdomains. These include aC-terminal transactivation function and a dimerization interface.Binding of the specific ligands to the nuclear receptor results in aconformation change that promotes interactions between thetransactivation domain and several transcriptional co-activatorcomplexes. These complexes destabilize chromatin and activatetranscription. Through this mechanism, nuclear receptors directlyregulate transcription in response to their specific ligands.

[0007] An important advance in the characterization of this superfamilyof regulatory proteins has been the discovery of a growing number ofgene products which possess the structural features of hormone receptorsbut which lack known ligands. These are known as orphan receptors, whichlike the classical members of the nuclear receptor superfamily, possessDNA and ligand binding domains. They are believed to be receptors foryet to be identified signaling molecules.

[0008] The peroxisome proliferator activated receptors (PPARs) representa subfamily of structurally related nuclear receptors. Three subtypeshave been described: PPARα, γ, and δ. The DNA recognition sequences forall PPAR subtypes are composed of a directly repeating core-siteseparated by 1 nucleotide. A common recognition sequence isXXXAGGTCAXAGGTCA (SEQ ID NO:1), however, the core-site (AGGTCA; SEQ IDNO:2) is variable and may change by one or more nucleotide. To bind DNA,PPARs must heterodimerize with the 9-cis retinoic acid receptor (RXR).

[0009] The α subtype of PPAR is expressed at high levels in liver andwas originally identified as a molecule that mediates thetranscriptional effects of drugs that induce peroxisome proliferation inrodents. In addition, PPARα binds to and regulates transcription of avariety of genes involved in fatty acid degradation (β- andω-oxidation). Mice lacking functional PPARα exhibit decreasedβ-oxidation capacity and are incapable of increasing this capacity inresponse to PPARα ligands). Further, these mice inappropriatelyaccumulate lipid in response to pharmacologic stimuli and developlate-onset obesity. Taken together, these data indicate that PPARαactsas both a sensor and an effector in a feedback loop that induces lipidcatabolism in response to fatty acid signals.

[0010] In contrast to PPARα, the γ subtype of PPAR plays a critical rolein the opposing process of fatty acid storage. PPARγ is expressed athigh levels in adipocytes where it has been shown to be critical foradipogenesis. Indeed, forced expression of PPARγ in fibroblastsinitiates a transcriptional cascade that leads to the expression ofadipocyte-specific genes and ultimately to triglyceride accumulation.This implies that signals which modulate PPARγ activity may serve aprimary role in regulatory energy homestasis.

[0011] PPARδ is ubiquitously expressed and binds several polyunsaturedfatty acids as well as carbaprostacyclin, a synthetic analog of PGI₂.PPARδ has been suggested to contribute to the control of embryoimplantation and the inhibitory effects of non-steroidalanti-inflammatory drugs on colon cancer.

[0012] That PPARs regulate lipid homeostasis implies that putative PPARligands represented endogenous regulators of lipid homeostasis. Oneligand for PPARγ has been identified 15-deoxy-Δ^(12,14)-prostaglandin J₂(15d-J₂). The thiazolidinedione class of anti-diabetic agents mimic15d-J₂, acting as potent ligands. Moreover, the potency ofthiazolidinediones as antidiabetic agents correlates with their in vitroaffinity for PPARγ. Forman et al., Cell 83:803-812 (1995); Wilson etal., J. Med. Chem. 39:665-668 (1996). These data suggest that PPARγmediates the antidiabetic activity of these compounds.

[0013] Several other studies have shown that thiazolidinedionessimultaneously promote insulin sensitization and increases in adiposecell number/mass in rodent models of NIDDM. Similarly, a geneticanalysis suggested a link between obesity and a lower degree of insulinresistance in humans harboring an activating mutation in the N-terminusPPARγ. Ristow et al., N. Engl. J. Med. 339:953-959 (1998). Thatactivation of PPARγ can induce adiopogenesis in cell culture as well aspromote insulin sensitization in vivo appears paradoxical given theepidemiological studies that link weight gain and obesity to NIDDM.However, like the pharmacologic data in rodents, this genetic datasuggests that PPARγ activation dissociates adipogenesis from insulinresistance.

[0014] Thiazolidinediones reverse insulin resistance in skeletal muscle,adipose tissue and hepatocytes. Komers and Vrana, Physiol. Res.47(4):215-225 (1998). An increase insulin responsiveness is accompaniedby a normalization of a wide range of metabolic abnormalities associatedwith NIDDM, including elevated levels of glucose, insulin,triglycerides, free fatty acids and LDL-cholesterol. Thiazolidinedionesdo not promote insulin secretion nor do they act as hypoglycemic agentsin non-diabetic animals, implying that PPARγ regulates genes thatreverse a critical step in the development of insulin resistance.

[0015] Several interesting hypotheses have been proposed to explain whatcauses insulin resistance and how PPARγ activation reverses thisprocess. Insulin resistance may result from increase in circulatinglevels of free fatty acids. McGarry, Science 258:766-770 (1992). If thisis the case, PPARγ activation would be predicted to reverse insulinresistance by promoting an increase in fatty acid storage in adipocyes.However, this does not account for the observation that free fatty acidsare not elevated in all diabetic models and that a lowering of fattyacids using other treatments is not sufficient to promote insulinsensitization. Alternatively, Spiegelman and colleagues have suggestedthat insulin resistance results from an increased production of TNFα inthe adipose tissue of diabetics. Uysal et al., Nature 389:610-614(1997). Under this theory, PPARγ ligands act by blocking theTNFα-mediated inhibition of insulin signaling, however this is notconsistent with all models of NIDDM. How PPARγ normalizes insulinresistance thus remains unclear.

[0016] PPARγ is expressed at high levels in both brown (BAT) and whiteadipose tissue (WAT). In vivo administration of PPARγ ligands have beenshown to increase the size of both fat depots. In principle, therefore,both of these tissues could be involved in regulating insulinresponsiveness. Transgenic mice with decreased levels of both BAT andWAT may be made by expressing the diphtheria toxin in these tissuesusing the adipocyte specific aP2 promoter. Burant et al., J. Clin.Invest. 100:2900-2908 (1997). By 8-10 months of age these miceapparently lack subcutaneous or intra-abdominal triglyceride-containingadipose tissue. The loss of triglyceride containing cells wasaccompanied by a progressive increase in insulin resistance and thedevelopment of diabetes. Despite the apparent loss of adipose tissue,administration of thiazolidinediones to these mice still resulted ininsulin sensitization. These findings indicate that the antidiabeticaction of thiazolidinedione occurs independently ofthiazolidinedione-induced increases in adipocyte triglyceride content,and perhaps independent of adipose tissue. Burant et al., J. Clin.Invest. 100:2900-2908 (1997). However, this may depend on how adipocyteis defined. It is known that PPARγ is induced early in the course ofadipogenesis and that PPARγ expression is required for subsequentactivation of the aP2 promoter in adipocytes. This transcriptionalcascade is followed by massive triglyceride accumulation. The strategyemployed by Graves and colleagues for generation of “fat-free” micedepends on expression of a toxic transgene under control of thefat-specific aP2 promoter. However, since the expression of the toxictransgene in fat requires the presence of PPARγ, these mice shouldpossess adipocyte-precursors which express PPARγ in the atrophicremnants of adipose tissue. Thus, it may be more precise to state thatthiazolidinedione action is independent of mature adipose tissue.Previous studies have not been designed to address the issue of whetherthe antidiabetic effects of thiazolidinediones are mediated by cells ofthe adipocyte lineage.

[0017] PPARγ may also be expressed in skeletal muscle and in the liverbut its level of expression is at least 10-fold lower in these tissuesthan in fat. The analysis of PPARγ expression in skeletal muscle hasbeen complicated by the presence of fat cells that interdigitate amongthe myocytes. Since PPARγ is expressed at high levels in fat, it ispossible that PPARγ transcripts seen on northern blots are derived fromthe contaminating fat cells. Immunohistochemical assays withPPARγ-specific antibodies have shown that PPARγ is expressed in myocytenuclei at low levels. Despite the ability of thiazolidinediones topromote glucose uptake sensitization of skeletal muscle in vivo, thesecompounds had no detectable effect on glucose uptake after a five-hourexposure. Since the antidiabetic effects of thiazolidinediones areobserved only after 1-2 weeks of treatment, a longer duration ofexposure may be required to elicit the antidiabetic response, however itis difficult to maintain phenotypic myocytes in culture for this lengthof time. For similar reasons, it is not clear whether the liver is adirect target for the antidiabetic effects of thiazolidinediones.

[0018] “Knockout” mice lacking the PPARγ gene have an embryonic lethalphenotype. Thus, these mice are not useful in studying the effects ofPPARγ in the adult animal. In principle, it might be possible to bypassthe embryonic lethal phenotype by generating tissue-specific knockoutsof PPARγ. In practice, this approach has been complicated by the need toexpress sufficient quantities of the cre-recombinase in the targettissue. Assuming these technical difficulties can be overcome, theresulting mice would be useful in an analysis of the physiologicalconsequences resulting from the loss of PPARγ function. In any case,these mice would not be useful to study the consequences of PPARγactivation. A method of studying what the effects would be in individualtissues upon activation of PPARγ by a drug, or the like would beenormously useful.

SUMMARY OF THE INVENTION

[0019] Accordingly, this invention provides a method of identifyingnuclear receptor controlled genes in a specific tissue of an animal,which comprises providing an expression vector containing aconstitutively active nuclear receptor gene which is fused at theN-terminus to the transcriptional activation domain of the Herpes viralVP16 protein, and is linked to a-promoter element which drivestissue-specific expression, transferring the constitutively activenuclear receptor gene to the animal, expressing the constitutivelyactive nuclear receptor gene in the animal, determining the level ofexpression of candidate target genes of the nuclear receptor in thetissue, and identifying genes which exhibit altered expression.

[0020] In another embodiment, the invention provides a method ofmodulating the in vivo expression of nuclear receptor controlled genesin a specific tissue of an animal, which comprises providing anexpression vector containing a constitutively active nuclear receptorgene which is fused at the N-terminus to the transcriptional activationdomain of the Herpes viral VP16 protein, and is linked to a promoterelement which drives tissue-specific expression, transferring theconstitutively active nuclear receptor gene to the animal, andexpressing the constitutively active nuclear receptor gene in vivo inthe animal.

[0021] In yet another embodiment, the invention provides a transgenicnon-human animal harboring a constitutively active nuclear receptor genewhich is fused at the N-terminus to the transcriptional activationdomain of the Herpes viral VP16 protein, and is linked to a promoterelement which drives tissue-specific expression. Preferably, the nuclearreceptor gene is PPARγ and the promoter element drives brown adiposetissue-specific expression. A preferred promoter is the promoter fromuncoupling protein 1.

[0022] In yet a further embodiment, the invention provides a method ofpreventing diabetes in a mammal having brown adipose tissue whichcomprises activity PPARγ expression specifically in brown adiposetissue.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a histogram depicting reporter-activity of PPARγ andVP-PPARγ.

[0024] FIGS. 2A-2D show which animals contain the indicated exogenousVP-PPARγ gene by RT-PCR using two primer pairs with confirmation bySouthern blot.

[0025]FIG. 3 presents RT-PCR analysis (3A; primer pair A, 740 bp; 3B:primer pair B, 679 bp) indicating that the VP-PPARγ1 transgene isspecifically expressed in brown fat.

[0026]FIG. 4 presents data on metabolic parameters in aged female wildtype (wt) mice and transgenic (Tg) mice expressing VP-PPARγ in brownfat.

[0027]FIG. 4A: fasting glucose;

[0028]FIG. 4B: insulin levels;

[0029]FIG. 4C: leptin levels;

[0030]FIG. 4D: serum triglycerides (mg/dL);

[0031]FIG. 4E: serum cholesterol (mg/dL);

[0032]FIG. 4F: food intake (g);

[0033]FIG. 4G: body weight (g);

[0034]FIG. 4H: daytime rectal temperature (°C.).

[0035]FIG. 5 shows brown adipose tissue from wild type and transgenicmice and demonstrates decreased steatosis in brown fat expressing theUCP-VPP-PPARγ transgene.

DETAILED DESCRIPTION OF THE INVENTION

[0036] Transgenic mice have been developed that express a constitutivelyactive form of PPARγ in either brown fat, white fat, skeletal muscle orliver. Each mouse line will be evaluated for parameters of insulinresponsiveness under normal and diabetogenic conditions. This will allowdetermination of whether activation of PPARγ target genes in any one (orcombination) of tissue(s) is sufficient to relieve insulin resistance invivo. As insulin resistance is a defining featured of NIDDM, a betterunderstanding of this phenomenon should aid in the development of moreeffective therapeutic strategies.

[0037] A constitutively active form of PPARγ was generated by fusing thetranscriptional activation domain of the Herpes viral VP16 protein tothe N-terminus of PPARγ (VP-PPARγ). N-terminus fusions were created asthis does not alter the DNA binding or dimerization activity ofreceptors. The VP16 transactivation domain was chosen because it is wellcharacterized and is known to function independent of nuclear receptorligands. Recent studies indicate that VP16 recruits a transcriptionactivation complex that is indistinguishable from that utilized by PPARγand other nuclear receptors (TRAP/SMCC). The altered receptor activatedtranscription via PPARγ response elements in the absence of ligand, yethas similar DNA binding and transactivation specificity as the wild-typereceptor. As shown in FIG. 1, the VP-PPARγ chimera activates PPARγresponse elements in the absence of ligand but has no effect onnon-PPARγ response elements including a closely related DR-1 typeresponse element (CRBPII) specific for RXR homodimers.

[0038] To further confirm the biological activity of VP-PPARγ, thisreceptor was tested for induction of adipocyte differentiation in theabsence of PPARγ ligands using the adipogenic system established byTontonoz and Spiegelman. Tontonoz and Spiegelman, Cell 79:1147-1156(1994). The NIH 3T3 cells in this system lack PPARγ and thus cannotdifferentiate into adipocytes. However, when infected withPPARγ-expressing retroviruses, these cells will undergo adipocytedifferentiation in the presence of PPARγ ligands. VP-PPARγ was clonedinto a replication defective retroviral expression vector (pBABE).Expression of the wild-type receptor in NIH-3T3 cells resulted in cellsthat underwent nearly 100% differentiation in response to PPARγ ligands.In contrast, the VP-PPARγ expressing retroviruses led to an equallyefficient adipocyte conversion in the absence of PPARγ ligand (data notshown). These results are consistent with the transfection studies andconfirm that VP-PPARγ acts as a constitutively active form of VP-PPARγin a biologically relevant system.

[0039] Transgenic expression vectors that would specifically directexpression of this chimera to the BAT, WAT, skeletal muscle and liver oftransgenic mice were then constructed. The transgenic expression vectorscontained VP-PPARγ linked to the following promoter elements which areknown to drive tissue-specific expression: creatine kinase in skeletalmuscle (Moller et al. Endocrinol. 137:2397-2405 (1996)); major urinaryprotein in liver (Held et al. EMBO J. 8:183-191 (1989)); aP2 in totaladipose tissue (Ross et al., Genes Dev. 7:1318-1324 (1993)); anduncoupling protein 1 in brown adipose tissue (Lowell et al., Nature336:740-742 (1993)). The tissue specificity of these promoters have beenwell documented and they are transcribed very late in fetal developmentor within a few weeks after birth. Thus, this method allows expressionof the chimeric receptor in a tissue and temporal-specific manner thatavoids the potential problem of developmental defects resulting fromtransgenic expression.

[0040] The transgenes described above were injected into zygotes derivedfrom C57BL/KsJ mice to create several lines of transgenic mice. Thesemice are known to be genetically susceptible to the development ofNIDDM. After birth, the transgenic mice were screened for integration ofthe transgene using two different sets of PCR primers. Positive micewere confirmed by Southern blot analysis using a VP16 probe thatspecifically recognizes the transgene. For each promoter construct,several founders were identified that have incorporated an apparentlyintact transgene (FIG. 2).

[0041] The founder mice were screened for tissue specific expression ofPPARγ-specific probes. Levels of expressed chimeric protein will bedetermined by western blot analysis using a monoclonal antibody (12CA5)that specifically recognizes a 9 amino acid epitope tag engineered intothe original VP-PPARγ chimera. Several lines containing intacttransgenes were further analyzed for tissue specific expression of thetransgene by RT-PCT analysis using two independent primer pairs thatspan the 5′ and 3′ end of the transgene. As indicated in FIG. 3, severallines expressed the transgene in brown fat, but not in other tissuescritical for glucose homeostasis, including white fat, skeletal musclesand liver.

[0042] To confirm the functionality of the transgene, levels ofexpression of several known PPARγ target genes (e.g., UCP, aP2) weremeasured. Elevated levels of expression of these target genes would beexpected in transgenic animals that express a functional VP-PPARγ.Different lines of mice expressing VP-PPARγ were selected for eachpromoter construct. These mice have been analyzed by RT-PCR and theresults indicate that the transgenes show the expected pattern ofexpression. For example, the mice containing the VP-PPARγ transgeneexpressed the transgene in brown fat but not in white fat, liver,skeletal muscle or other tissues that were examined. Colonies of themice were expanded for the analyses described below.

[0043] A cursory phenotypic analysis of the mice suggests that thetransgenes are appropriately expressed. Specifically, the UCP-VP-PPARγmice would be expected to be expressed uniquely in brown fat and to tendto an overproduction of uncoupling protein 1. Tai et al., J. Biol. Chem.271:29909-29914 (1996). As UCP-1 activity is associated with the burningof fat, these mice might be expected to have decreased white adiposestores.

[0044] To examine the effect of VP-PPARγ expression in brown fat on thedevelopment of diabetes and insulin resistance, the metabolic effects oftransgene expression in old mice (1-1.5 years) that spontaneouslydevelop diabetes was examined. As shown in FIG. 4, wild-type female micehad elevated fasting glucose (FIG. 4A) and insulin levels (FIG. 4B)whereas their transgenic counterparts had circulating glucose values inthe normal range. These data indicate that the VP-PPARγ expression inbrown fat prevents the development of insulin resistance and diabetes.Transgenic mice also displayed lower leptin levels (FIG. 4C) but showedno differences in the levels of serum triglycerides (FIG. 4D) andcholesterol (FIG. 4E). Examination of body weight (FIG. 4F) and foodintake (FIG. 4G) indicated that food intake is higher in the transgenicmice than in wild-type controls. However, despite the increased foodintake, these mice did not show an elevated body weight, suggesting thattheir metabolism is more efficient. Transgenic mice had elevated bodytemperatures, suggesting that the transgene promotes increased energyconsumption via thermogenesis (FIG. 4H).

[0045] Histologic analysis of brown fat from wild-type and transgenicmice indicated that transgenic mice have smaller fat cells withdecreased accumulation of triglyceride (FIG. 5). Brown fat wascollected, sectioned and stained with Oil-Red-O, a triglyceride specificdye. The appearance of the transgenic brown fat was remarkably similarto that of brown fat from younger mice. These histological features areconsistent with improved function of brown adipose tissue (increasedthermogenesis) in the transgenic mice.

[0046] The data presented above indicate that activation of PPARγ inbrown fat (or UCP1-expressing cells) is sufficient to prevent thedevelopment of diabetes. Therefore, tissue-specific drugs such as TZDanalogs which specifically activate PPARγ in brown fat can act asspecific antidiabetic agents, free of the other systematic effects ofPPARγ activation. Moreover, the invention disclosed here provides arational justification as well as critical reagents for a screen toidentify this putative regulator of insulin responsiveness. Genetherapies, such as that described above, in which PPARγ is specificallyactivated in brown adipose tissue, may be used to prevent or treatdiabetes.

[0047] The data disclosed above show that the antidiabetic effects ofTZDs/PPARγ can be dissociated from the deleterious effects of enhancedwhite adipose mass which are known to result from TZDS. Moreover,dissociation of the insulin sensitizing effects of PPARγ from its lipidlowering effects implies that elevated triglyceride levels are not anunderlying cause of NIDDM. The data presented here also indicate thatPPARγ can promote insulin sensitization independent of a direct effecton resistin or leptin genes. However, PPARγ activation in brown fat canproduce an indirect compensatory effect on white adipose tissue, sinceleptin levels were lower in UCP1-VP-PPARγ mice compared to wild type.

1 2 1 16 DNA mammalian misc_feature (1)..(1) n = any nucleotide base 1nnnaggtcan aggtca 16 2 6 DNA mammalian 2 aggtca 6

1. A transgenic non-human animal, the genome of which comprises a genethat encodes a constitutively active nuclear receptor which is fused atthe N-terminus to the transcriptional activation domain of the Herpesviral VP16 protein, wherein said gene encoding said nuclear receptor islinked to a promoter element which drives tissue-specific expression. 2.A transgenic animal of claim 1 wherein said nuclear receptor gene isPPARγ.
 3. A transgenic animal of claim 1 wherein said promoter elementdrives expression specifically in uncoupling protein 1-expressing cells.4. A transgenic animal of claim 1 wherein said promoter element is thepromoter for uncoupling protein 1.